The packed-bed adsorption and elution of aqueous solutions of whey concentrate powders were investigated at pH 3.7 using a 5-mL SP Sepharose FF column to separate and isolate two major proteins namely, alpha-lactalbumin (ALA) and beta-lactoglobulin (BLG) from these solutions. ALA displaced and eluted BLG from the column its a pure form. Pure ALA could then be eluted with good recovery. A novel consecutive two-stage separation process was developed to separate ALA and BLG front whey concentrate mixtures. Almost all of the BLG in the feed was recovered, with 78% being recovered at 95% purity and a further 20% at 86% purity. In addition, 67% of ALA was recovered, 48% at 54% purity and 19% at 60% purity. (C) 2009 American Institute of Chemical Engineers Biotechnol. Prog., 26: 192-199, 2010